CN114874963B - Recombinant vibrio natriegens for producing hydroxytyrosol and application thereof - Google Patents
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Abstract
The invention relates to the technical field of bioengineering, in particular to recombinant vibrio natriegens for producing hydroxytyrosol and application thereof. The invention provides a new application of vibrio natriegens in producing hydroxytyrosol. The engineered bacteria of vibrio natriegens obtained by modification can be used for whole-cell catalytic synthesis of hydroxytyrosol, can efficiently promote accumulation of hydroxytyrosol, and can also be used as Chassis bacteria for constructing a hydroxytyrosol production strain. The method for producing hydroxytyrosol based on vibrio natriegens provided by the invention can shorten the production period of hydroxytyrosol, improve the yield of hydroxytyrosol and reduce the difficulty in removing downstream endotoxin while ensuring the yield of hydroxytyrosol.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to recombinant vibrio natriegens for producing hydroxytyrosol and application thereof.
Background
Hydroxytyrosol (HT) is a natural polyphenolic compound with chemical name of 3, 4-dihydroxyphenylethanol and molecular formula of C 4 H 10 O 3 The relative molecular mass was 154.16.
The hydroxytyrosol is a light yellow oily liquid, belongs to an amphiphilic molecule, and has good water solubility and fat solubility. Epidemiological investigation found that: the morbidity and mortality of cardiovascular diseases, cancers and the like of residents in the Mediterranean countries who eat olive oil for a long time are low; this health effect is closely related to hydroxytyrosol, a polyphenolic compound rich in olive oil. Hydroxytyrosol, which is a polyphenol compound, has good oxidation resistance, and is found to have a treatment effect on oxidative stress diseases, nervous system diseases, cardiovascular diseases, metabolic syndrome, inflammation, tumors and other diseases. In addition, the hydroxytyrosol has wide application due to the special biological and pharmacological activity: in the food industry, the product can be used as a natural preservative to prevent the oxidative deterioration of foods; in the field of nutrition and health care, can be used as a nutritional supplement and an antibacterial agent and added into various foods.
Currently, methods for producing hydroxytyrosol are chemical synthesis and plant extraction. The chemical synthesis method of hydroxytyrosol is complex, the toxicity of the related organic reagents is high, the environmental pollution is serious, and the price of the used catalyst is high.
In the prior art, most of hosts used for biosynthesis of hydroxytyrosol are escherichia coli, for example: the patent application CN110616180A discloses that L-phenylalanine dehydrogenase, alpha-keto acid decarboxylase and alcohol dehydrogenase genes are introduced into escherichia coli expression genetic engineering bacteria, and L-dopa is used as a substrate to catalyze and synthesize hydroxytyrosol. Currently, the yield of biosynthetic hydroxytyrosol still needs to be improved.
Disclosure of Invention
The invention aims to provide a recombinant vibrio natriegens for producing hydroxytyrosol and application thereof. Another object of the present invention is to provide a method for producing hydroxytyrosol by using Vibrio natriegens.
The biosynthesis of different biochemicals often requires the selection of a suitable microbial platform, for example: although escherichia coli is the most common microbial platform, due to the distribution characteristics of intracellular metabolic flux and the characteristics of overactive catabolism of certain substances, the escherichia coli is suitable for biosynthesis of some biochemicals, but some biochemicals are not suitable for biosynthesis by using the escherichia coli. Regarding the biosynthesis of hydroxytyrosol, almost all the prior art is carried out by using Escherichia coli, and no report on the production of hydroxytyrosol by using Vibrio natriegens (Vibrio natriegens) is found. At present, the degradation and regulation mechanism of the hydroxytyrosol in the vibrio natriegens is not clear, and the inventor finds that the vibrio natriegens have very strong degradation capability on the hydroxytyrosol in the earlier development process, which may also be the reason that the vibrio natriegens are not adopted to produce the hydroxytyrosol at present. However, in the subsequent studies, the inventors unexpectedly found that the vibrio natriegens having strong degradation ability to hydroxytyrosol can also efficiently accumulate hydroxytyrosol, and the ability of catalyzing the synthesis of hydroxytyrosol is even higher than that of escherichia coli. The present invention has been further completed based on the above findings.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides the use of Vibrio natriegens in the production of hydroxytyrosol.
Preferably, the application is whole-cell catalytic production of hydroxytyrosol using vibrio natriegens.
Preferably, the use comprises the step of expressing the enzyme for the synthesis of hydroxytyrosol by vibrio natriegens.
Preferably, the applications include expressing the enzyme for the synthesis of hydroxytyrosol and for catalyzing NAD by Vibrio natriensis + A step of synthesizing NADH enzyme.
The above-mentioned enzymes for synthesizing hydroxytyrosol include, but are not limited to, enzymes catalyzing the synthesis of hydroxytyrosol from tyrosol, enzymes catalyzing the synthesis of hydroxytyrosol from tyrosine, and the like.
Wherein the enzyme catalyzing tyrosol to hydroxytyrosol comprises 4-hydroxyphenylacetic acid 3-monooxygenase.
The above-described catalytic NAD + Enzymes that synthesize NADH include, but are not limited to, formate dehydrogenase, glucose dehydrogenase, and the like.
Further preferably, the application comprises: introducing a nucleic acid molecule comprising coding genes for 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase into Vibrio natriensis to obtain recombinant Vibrio natriensis, and producing hydroxytyrosol by using the recombinant Vibrio natriensis.
As for 4-hydroxyphenylacetic acid 3-monooxygenase and formate dehydrogenase, the invention discovers that for vibrio natriegens, 4-hydroxyphenylacetic acid 3-monooxygenase derived from escherichia coli and formate dehydrogenase derived from mycobacterium intracellulare can be better expressed in vibrio natriegens, and is beneficial to promoting the efficient synthesis of hydroxytyrosol.
Preferably, the 4-hydroxyphenylacetic acid 3-monooxygenase as described above is derived from E.coli. The formate dehydrogenase is derived from M.intracellulare.
In some embodiments of the invention, the 4-hydroxyphenylacetate 3-monooxygenase is a protein of accession No. WP _000801472.1 and WP _001175451.1 and the formate dehydrogenase is a protein of accession No. WP _009957650.1.
As for the genes encoding 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase, those skilled in the art can obtain them from the amino acid sequences of 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase. The nucleotide sequence of the gene encoding the above-mentioned protein is not unique based on the codon rule, but all genes capable of encoding 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase are within the scope of the present invention.
In the application, the production of hydroxytyrosol by utilizing the recombinant vibrio natriegens is specifically that tyrosol is used as a substrate, and the recombinant vibrio natriegens are used for whole-cell catalysis.
Preferably, the application comprises: culturing the recombinant vibrio natriegens, inducing the expression of 4-hydroxyphenylacetic acid 3-monooxygenase and formate dehydrogenase, collecting thalli, and carrying out whole-cell catalysis by using tyrosol as a substrate.
In a second aspect, the present invention provides a recombinant Vibrio natriegens that express a 4-hydroxyphenylacetate 3-monooxygenase and a formate dehydrogenase.
Preferably, the 4-hydroxyphenylacetate 3-monooxygenase is derived from E.coli, and/or the formate dehydrogenase is derived from M.intracellulare.
Further preferably, the 4-hydroxyphenylacetic acid 3-monooxygenase is WP _000801472.1 and WP _001175451.1, and/or the formate dehydrogenase is WP _009957650.1.
The invention finds that the expression of the 4-hydroxyphenylacetate 3-monooxygenase and the formate dehydrogenase is preferably induced, and the induction by the arabinose is more favorable for the Vibrio natriegens to express the 4-hydroxyphenylacetate 3-monooxygenase and the formate dehydrogenase.
Preferably, the expression of the 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase initiates transcription from the arabinose promoter.
Further preferably, vibrio natriegens are caused to express 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase by introducing into the Vibrio natriegens expression plasmids containing genes encoding 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase.
In some embodiments of the present invention, the expression plasmid introduced into Vibrio natriegens comprises an arabinose promoter and a 4-hydroxyphenylacetate 3-monooxygenase-encoding gene and a formate dehydrogenase-encoding gene.
In some embodiments of the invention, the expression plasmid is constructed from the plasmid petdue.
In some embodiments of the invention, the arabinose promoter, the 4-hydroxyphenylacetate 3-monooxygenase-encoding gene, and the formate dehydrogenase-encoding gene are linked in sequence in the expression plasmid.
In some embodiments of the invention, the nucleotide sequence of the arabinose promoter is as shown in SEQ ID No. 1.
In some embodiments of the invention, the starting strain for the recombinant vibrio natriegens construction is vibrio natriegens Vmax.
In a third aspect, the present invention provides the use of the recombinant Vibrio natriegens described above in the production of hydroxytyrosol.
In a fourth aspect, the present invention provides a method for producing hydroxytyrosol, which comprises the step of culturing the recombinant Vibrio natriegens described above.
Specifically, the method comprises the following steps: culturing the recombinant vibrio natriegens, inducing the expression of 4-hydroxyphenylacetic acid 3-monooxygenase and formate dehydrogenase by an inducer, collecting thalli, and producing hydroxytyrosol by whole-cell catalysis by using the collected thalli and using tyrosol as a substrate.
Preferably, the induction is at OD 600 When the expression level is 0.3-0.8, arabinose is used to induce the expression of 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase.
Further preferably, the induction is carried out at 32-37 ℃; the inducer is 0.1-1% of L-arabinose.
Preferably, the cells are collected after induction for 4 to 24 hours (preferably 4 to 8 hours), the collected cells are resuspended in a catalytic reaction solution, and whole-cell catalysis is carried out at 16 to 37 ℃ (preferably 30 to 32 ℃).
Wherein the catalytic reaction liquid comprises the following components: 5-8 g/L of disodium hydrogen phosphate, 2-4 g/L of potassium dihydrogen phosphate, 0.3-0.8 g/L of sodium chloride, 0.8-1.2 g/L of ammonium chloride, 2-2.5 g/L of sodium formate and 2-8 g/L of tyrosol.
The time for whole-cell catalysis is not particularly limited in the present invention, and the time for terminating catalysis can be determined by monitoring the production of hydroxytyrosol.
In some embodiments of the invention, whole cells catalyze from 2 to 48 hours.
The invention has the beneficial effects that: the invention provides a new application of vibrio natriegens in producing hydroxytyrosol. The engineered bacteria of vibrio natriegens obtained by modification can be used for whole-cell catalytic synthesis of hydroxytyrosol, can efficiently promote accumulation of hydroxytyrosol, and can also be used as Chassis bacteria for constructing a hydroxytyrosol production strain. In addition, compared with Escherichia coli, the growth speed of vibrio natriegens is higher, the fermentation period can be shortened, and the production rate is improved; the endotoxin level is low, and the method is more suitable for producing related natural compound products in the fields of medicine, nutrition and food.
The method for producing hydroxytyrosol based on vibrio natriegens provided by the invention can shorten the production period of hydroxytyrosol, improve the yield of hydroxytyrosol and reduce the difficulty in removing downstream endotoxin while ensuring the yield of hydroxytyrosol.
Drawings
FIG. 1 is a graph showing the comparison between Vmax-HT of recombinant Vibrio natriensis and E.coli-HT of recombinant E.coli in example 2 of the present invention.
FIG. 2 is a graph showing the comparison of hydroxytyrosol degradation abilities of the recombinant Vibrio natriegens Vmax-HT and the recombinant Escherichia coli E.
FIG. 3 is a graph showing the comparison of hydroxytyrosol synthesizing abilities of the recombinant Vibrio natriegens Vmax-HT and the recombinant Escherichia coli E.coli-HT of the present invention in example 4.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 construction of recombinant Vibrio natriegens Vmax-HT and E.coli E
1. The sequences of the target genes HpaB (NCBI accession No.: WP-000801472.1) and HpaC (NCBI accession No.: WP-001175451.1) derived from Escherichia coli, and the sequence of the target genes HpaB (HpaC) and FDH (NCBI accession No.: WP-009957650.1) derived from M.intracellulare were downloaded from the NCBI database and gene synthesis was performed.
2. The synthetic gene was amplified by cloning, and the fragment was inserted between SacI and HindIII in plasmid pETDuet using Gibson assembly method to obtain plasmid pETDuet-HpaB-HpaC-FDH.
3. The arabinose promoter sequence (SEQ ID NO. 1) is cloned and amplified, the part except the T7 promoter in the plasmid pETDuet-HpaB-HpaC-FDH is cloned and amplified, and two pieces obtained by amplification are spliced by a Gibson assembly method to obtain the plasmid pETDuet-pBAD-HpaB-HpaC-FDH.
4. coli-HT, wherein the competence of escherichia coli BL21 (DE 3) used for transformation was commercially competent (purchased from transcgen Biotech co., ltd).
5. The recombinant plasmid pETDuet-pBAD-HpaB-HpaC-FDH is introduced into the Vmax of Vibrio natriensis to obtain a corresponding recombinant strain Vmax-HT, wherein the Vmax of Vibrio natriensis used for transformation is competent for commercial use (purchased from Codex DNA, inc).
Example 2 analysis of growth of recombinant Vibrio natriegens Vmax-HT and recombinant E.coli E
1. The recombinant strains Vmax-HT and E.coli-HT constructed in example 1 were removed from glycerol tubes at-80 ℃ and streaked out on LB solid plates and cultured overnight at 37 ℃. Subsequently, single colonies were picked from the plate and inoculated into 3ml of LBv2 (peptone 10g/L, yeast extract 5g/L, sodium chloride 11.92g/L, potassium chloride 0.313g/L, magnesium chloride 2.203 g/L) and LB (peptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L) liquid medium, respectively, and cultured at 37 ℃ and 220rpm for activation.
2. Recombinant strains Vmax-HT, EThe HT-activated bacterial liquid was inoculated into 20mL LBv2 and LB media, respectively, and the initial OD was controlled 600 The cells were cultured at 37 ℃ in a shaking flask of 220rpm and 150mL. Samples were then taken at different time points and OD was determined 600 The value is obtained.
OD obtained with different time points 600 Values were plotted against growth curves.
The results are shown in FIG. 1: in the initial growth stage (0-7 hours), the recombinant vibrio natriegens Vmax-HT has obvious growth advantages compared with the strain E.coli-HT, and simultaneously has higher biomass all the time in the whole measurement period of 24 hours, which shows that compared with the strain E.coli-HT, the recombinant vibrio natriegens Vmax-HT has higher growth rate and higher biomass, and can shorten the fermentation period in industrial application.
Example 3 analysis of hydroxytyrosol degradation Capacity of recombinant Vibrio natriensis Vmax-HT and recombinant Escherichia coli E
1. Recombinant strains Vmax-HT, e.coli-HT were activated as described in example 2.
2. Respectively inoculating the activated bacterial liquid of the recombinant strains Vmax-HT and E.coli-HT to 40mL LBv2 and LB culture medium according to 1%, culturing at 37 ℃ and 220rpm for 16 hours, and determining OD 600 The value is obtained.
3. According to the OD of the bacterial liquid 600 Calculating the value, respectively taking appropriate amount of bacterial liquid, centrifuging at 4000rpm for 10min, collecting thallus, making two bacterial strains have equal thallus amount, and resuspending thallus with 20mL reaction system to make final OD 600 All equal to 4, with cell-free reaction as control, incubated at 30 ℃ and 220 rpm. The reaction system comprises: 6.8g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 0.5g/L sodium chloride, 1g/L ammonium chloride and 0.67g/L hydroxytyrosol.
4. Samples were taken at various time points to determine the amount of hydroxytyrosol remaining therein.
The results are shown in FIG. 2: with the prolongation of the incubation time, the contents of hydroxytyrosol in the recombinant vibrio natriegens, escherichia coli and cell-free experimental groups are all reduced, wherein the degradation level of the hydroxytyrosol in the recombinant escherichia coli experimental groups is equivalent to that of the hydroxytyrosol under the cell-free condition, and the recombinant vibrio natriegens show obviously stronger hydroxytyrosol degradation capability.
Example 4 detection of Hydroxytyrosol synthesizing Capacity of recombinant Vibrio natriensis Vmax-HT and recombinant Escherichia coli E
1. Recombinant strains Vmax-HT, e.coli-HT were activated as described in example 2.
2. The bacterial liquid after activation of the recombinant strains Vmax-HT and E.coli-HT is respectively inoculated into 20mL LBv2 and LB culture medium according to 1 percent, and cultured in shaking flasks of 220rpm and 250mL at 37 ℃. Two recombinant strains OD 600 When 0.5 was reached, L-arabinose was added to the medium at a final concentration of 0.5% for induction.
3. Centrifuging the bacteria liquid for 10min at 4000rpm after 6 hours of induction to collect thalli, resuspending the thalli by using a 20mL reaction system, and carrying out whole-cell catalysis at 30 ℃ and 220rpm, wherein the reaction system comprises: 6.8g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 0.5g/L sodium chloride, 1g/L ammonium chloride, 2.04g/L sodium formate and 4.145g/L tyrosol.
4. Samples were taken 4 hours after whole cell catalysis to determine hydroxytyrosol production.
The results are shown in table 1 and fig. 3: compared with the strain E.coli-HT, the recombinant vibrio natriegens Vmax-HT has higher hydroxytyrosol yield, the required fermentation period is shorter, and the recombinant vibrio natriegens Vmax-HT has higher hydroxytyrosol yield.
TABLE 1 comparison of yield, cycle and yield of hydroxytyrosol synthesized by recombinant Vibrio natriegens Vmax-HT and recombinant Escherichia coli E
The results of the above examples show that the recombinant Vibrio natriegens Vmax-HT has stronger hydroxytyrosol degradation capability than the existing recombinant Escherichia coli E.coli-HT, but the hydroxytyrosol synthesis capability, yield and production rate of the recombinant Vibrio natriegens Vmax-HT are obviously stronger than those of the Escherichia coli E.coli-HT, and meanwhile, the recombinant Vibrio natriegens Vmax-HT has faster growth speed and low endotoxin level, so that the Vibrio natriegens have the potential to become better Chassis bacteria for synthesizing hydroxytyrosol.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (7)
1. Use of vibrio natriegens for the production of hydroxytyrosol, characterized in that the use comprises: introducing a nucleic acid molecule comprising coding genes of 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase into Vibrio natriensis to obtain recombinant Vibrio natriensis, and producing hydroxytyrosol by using the recombinant Vibrio natriensis;
the 4-hydroxyphenylacetic acid 3-monooxygenase is WP _000801472.1 and WP _001175451.1, and the formate dehydrogenase is WP _009957650.1.
2. The use of claim 1, wherein the production is whole cell catalysis using the recombinant vibrio natriegens using tyrosol as a substrate.
3. A method for producing hydroxytyrosol, which comprises the steps of culturing recombinant vibrio natriegens;
the recombinant vibrio natriegens express 4-hydroxyphenylacetic acid 3-monooxygenase and formate dehydrogenase;
the 4-hydroxyphenylacetic acid 3-monooxygenase is WP _000801472.1 and WP _001175451.1, and the formate dehydrogenase is WP _009957650.1.
4. The method of claim 3, wherein the expression of 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase initiates transcription from an arabinose promoter.
5. Process for the production of hydroxytyrosol as claimed in claim 3 or 4, which process comprises: culturing the recombinant vibrio natriegens, inducing the expression of 4-hydroxyphenylacetic acid 3-monooxygenase and formate dehydrogenase by an inducer, collecting thalli, and producing hydroxytyrosol by whole-cell catalysis by using the collected thalli and using tyrosol as a substrate.
6. The method of claim 5, wherein the induction is at OD 600 When the expression level is 0.3-0.8, arabinose is used to induce the expression of 4-hydroxyphenylacetate 3-monooxygenase and formate dehydrogenase.
7. The method of claim 6, wherein the cells are collected after the induction for 4 to 24 hours, resuspended in a catalytic reaction solution, and subjected to whole-cell catalysis at 16 to 37 ℃;
the catalytic reaction liquid comprises the following components: 5-8 g/L of disodium hydrogen phosphate, 2-4 g/L of potassium dihydrogen phosphate, 0.3-0.8 g/L of sodium chloride, 0.8-1.2 g/L of ammonium chloride, 2-2.5 g/L of sodium formate and 2-8 g/L of tyrosol.
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